Quantification of intracellular cAMP and IP1 was performed using the HTRF-cAMP dynamic kit and the HTRF-IP1 kit, respectively, on a Mithras LB 940 reader (Berthold Technologies) according to the manufacturer's instructions37 (link). For cAMP assays with M2-CHO cells, the Gi-biased muscarinic agonist iper-6-phth (ref. 30 (link)) was applied.
For washout experiments, cells were pre-incubated for 1 h with FR (1 μM) and then washed three times for 5 min with 750 μl PBS, resuspended in fresh stimulation buffer, and seeded into a 384-well plate. For IP1 experiments with Gα14 and Gα16 (UMR cDNA resource center), HEK293 cells were transiently co-transfected with ORL1 cDNA using FuGENE HD transfection reagent (Promega).
For washout experiments, cells were pre-incubated for 1 h with FR (1 μM) and then washed three times for 5 min with 750 μl PBS, resuspended in fresh stimulation buffer, and seeded into a 384-well plate. For IP1 experiments with Gα14 and Gα16 (UMR cDNA resource center), HEK293 cells were transiently co-transfected with ORL1 cDNA using FuGENE HD transfection reagent (Promega).
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